MOLECULAR-CLONING AND CHARACTERIZATION OF A NOVEL FORM OF NEUROPEPTIDE GENE AS A DEVELOPMENTALLY-REGULATED MOLECULE

To examine the molecular basis controlling neuronal differentiation, s ubtraction library construction and differential screening were used t o identify cDNAs whose mRNA levels are regulated in mouse NS20Y cells by dibutyryl cyclic AMP treatment, One of them, N27K, whose mRNA incre ases transiently during both neuronal differentiation in NS20Y cells a nd development in mouse brain. The deduced amino acid sequence of N27K comprises 212 amino acid residues and is a novel form of a precursor protein for a new neuropeptide nociceptin/ orphanin FQ, which we indep endently cloned as N23K. That is, the putative protein encoded by N27K is 25 amino acids longer than that encoded by N23R, Using an antibody against a C-terminal peptide of the N27K: protein that recognizes a 2 7-kDa protein in Western blot analysis, a punctate structure in the pe rinuclear region and areas near the tip of neurites is visualized in n eurally differentiating NS20Y cells, The time of maximal expression co rrelates with periods of neurite extension, and expression decreases a s the neuritic network develops. Immunohistochemistry of tissue sectio ns of the mouse central nervous system revealed that reactivity for th e anti-N27K protein antibody can detected in early generated neurons a t embryonic day 14, in virtually all immature neurons at postnatal day 1, and in subsets of neurons of discrete brain regions such as the hy pothalamus and spinal cord in adults. This remarkable redistribution s uggests that N27K may be involved in a process in neurite outgrowth an d nervous system development.